Heidi K. Goethert

Meningoencephalitis from Borrelia miyamotoi in an Immunocompromised Patient

Ixodes ticks serve as vectors for Borrelia burgdorferi, the agent of Lyme disease. Globally, these ticks often concurrently harbor B. miyamotoi, a spirochete that is classified within the relapsing-fever group of spirochetes. Although humans presumably are exposed to B. miyamotoi, there are limited data suggesting disease attributable to it. We report a case of progressive mental deterioration in an older, immunocompromised patient, and even though Kochs postulates were not met, we posit B. miyamotoi as the cause, owing to its direct detection in cerebrospinal fluid (CSF) with the use of microscopy and a polymerase-chain-reaction (PCR) assay. It is likely that B. miyamotoi is an underrecognized cause of disease, especially in sites where Lyme disease is endemic.

Entomologic and Serologic Evidence of Zoonotic Transmission of Babesia microti, Eastern Switzerland

We evaluated human risk for infection with Babesia microti at a site in eastern Switzerland where several B. microti–infected nymphal Ixodes ricinus ticks had been found. DNA from pooled nymphal ticks amplified by polymerase chain reaction was highly homologous to published B. microti sequences. More ticks carried babesial infection in the lower portion of the rectangular 0.7-ha grid than in the upper (11% vs. 0.8%). In addition, we measured seroprevalence of immunoglobulin (Ig) G antibodies against B. microti antigen in nearby residents. Serum from 1.5% of the 396 human residents of the region reacted to B. microti antigen (>1:64), as determined by indirect immunofluorescence assay (IgG). These observations constitute the first report demonstrating B. microti in a human-biting vector, associated with evidence of human exposure to this agent in a European site.

Borrelia miyamotoi Disease in the Northeastern United States: A Case Series

Context The first known cases of Borrelia miyamotoi infection in North America were reported in 2013. The incidence, prevalence, and clinical spectrum of this infection are currently undefined. Contribution A review of the clinical records of 51 of 97 patients with acute B. miyamotoi infection diagnosed by polymerase chain reaction testing at a reference laboratory provides preliminary data on the clinical presentation, course, and response to antibiotics of this infection. Caution The study was not prospective or population-based. Implication Borrelia miyamotoi disease may be an emerging tickborne infection of clinical significance in the northeastern United States. The public health burden of infectious agents transmitted by the deer tick (Ixodes dammini, also known as the blacklegged tick or I. scapularis), comprising Borrelia burgdorferi, Babesia microti, Anaplasma phagocytophilum, and deer tick virus (Powassan virus lineage II), seems to be increasing in the northeastern United States (14). The first human cases of infection with Borrelia miyamotoi, previously considered nonpathogenic, were identified in Russia and described in 2011 (5, 6). In 2013, the American index case of B. miyamotoi disease (BMD) was described (7), and 2 other case patients (8) were reported with a clinical illness that was initially diagnosed as human anaplasmosis. Cases have subsequently been identified in New England (9), the Netherlands (10), and Japan (11). The prevalence of B. miyamotoi in host-seeking deer ticks (about 1% to 5% [1214]) is such that human exposure is likely; indeed, a serosurvey suggests that 10% of tick-exposed New England residents may have been exposed to B. miyamotoi (15). It is possible that zoonotic transmission has been common but not discriminated from other deer ticktransmitted zoonoses because the acute clinical presentation of BMD cases reported to date is nonspecific. To better define the clinical spectrum of this newly recognized zoonosis, we identified definitive cases of acute BMD by polymerase chain reaction (PCR) analysis of patient blood samples sent to a reference laboratory by clinical practices. We summarized the clinical features of BMD from 51 case patients out of 97 whom we identified as having active B. miyamotoi infection. In addition, we describe relevant associated laboratory findings and compare the observed frequency of BMD with that of human anaplasmosis and babesiosis, thereby providing a preliminary assessment of its relative public health burden. Methods Blood Samples Emergency departments, urgent care clinics, and primary care offices in Massachusetts, Rhode Island, New Jersey, and New York sent whole blood samples (EDTA-anticoagulated) to IMUGEN (Norwood, Massachusetts) as part of the clinical management of patients who were acutely symptomatic with features or laboratory findings suggestive of possible tickborne infection (typically fever, myalgia, flu-like illness, headache, or rash). An active detection protocol was adopted to identify samples that contained evidence of B. miyamotoi DNA during the 2013 and 2014 transmission seasons (1 April to 30 November). A positive B. miyamotoi finding on PCR prompted a request for chart review, and we focused on patients under our care with a goal of obtaining detailed clinical histories on at least 50 patients. The study was approved by the New England Institutional Review Board, and written, informed consent was obtained for clinical and laboratory follow-up of patients with positive PCR findings for Borrelia species. Molecular Detection of Infection Specimen receipt and handling, DNA extraction, and PCR setup were performed with enhanced contamination control practices and precautions at IMUGEN, which operates under good laboratory practice standards, Clinical Laboratory Improvement Amendments and College of American Pathologists certification, and New York State approval (Clinical Laboratory Evaluation Program). The laboratory has environmentally separate specimen processing, archive, and molecular extraction and setup areas. Extracted DNA from whole blood was tested in triplicate for the presence of Borrelia species by real-time PCR (Appendix) (16). Real-time PCR targeting the msp2 gene of A. phagocytophilum and the 18S ribosomal RNA gene of B. microti was performed from whole blood DNA extractions as previously described (1720). Representative samples were independently analyzed by PCR sequencing at the Telford laboratory of Tufts University (Appendix). B. burgdorferi Serology Sera were tested at IMUGEN by antibody capture enzyme immunoassay (EIA) for IgM, IgA, and IgG isotypes to B. burgdorferi sensu stricto strain 49736 and by immunoblot for IgM and IgG isotypes to B. burgdorferi sensu stricto strain G39/40 (2123). Immunoblot findings were interpreted according to the 2-tiered protocol (24). Recombinant B. miyamotoi GlpQ Enzyme-Linked Immunosorbent Assay The glycerophosphodiester phosphodiesterase (GlpQ) antigen has been found to be useful in the serodiagnosis of relapsing fever (25, 26). Given the genetic relatedness of B. miyamotoi to relapsing fever spirochetes, we analyzed the utility of GlpQ for confirmation of a BMD diagnosis. The GlpQ gene sequence from B. miyamotoi (GenBank accession number AY368276) was used as the basis for cloning and expression as a 38-kDa recombinant protein (rGlpQ), which was used in an indirect EIA for detection of antibody to B. miyamotoi (Appendix). Case Definitions A positive blood sample on 2 independent PCR assays (genus- and species-specific) was considered definitive evidence of active B. burgdorferi or B. miyamotoi infection. A blood sample that was repeatedly positive (reextracted and retested from the original sample) for B. microti or A. phagocytophilum on PCR was considered definitive evidence of active infection. Role of the Funding Source Internal funding from IMUGEN enabled the development and validation of test methods, the study design and execution, the clinical analysis, and the laboratory studies. The decision to prepare a manuscript for publication was not predicated on the availability of funding or the funding source. Results Confirmation of B. miyamotoi as the Infecting Agent We attempted to sequence portions of the GlpQ and flagellin genes, as previously described (7), from the 23 BMD cases from 2013 for which we had adequate remaining blood samples for DNA extraction. The organism was confirmed to be B. miyamotoi (GenBank accession numbers KP754938 to KP754960) in 17 samples (GlpQ, fla, or both sequences were obtained from the sample). The other 6 samples contained few spirochetes; those with high cycle threshold values in the real-time GlpQ PCR did not provide sequence (Spearman rank correlation coefficient, 0.67 [P< 0.001]). Frequency of BMD Between 1 April 2013 and 31 October 2014, we engaged in active case detection using specimens submitted to IMUGEN and identified 97 patients whose samples contained B. miyamotoi DNA. Of these, we obtained clinical information on 51 who had not been previously reported (7, 8) from selected practices in Massachusetts, Rhode Island, New Jersey, and New York. The months of onset or diagnosis of these BMD cases (Figure 1) overlapped with those for the other deer ticktransmitted infections, although most cases had onset in July and August. Figure 1. Seasonal distribution of acute Borrelia miyamotoi incident infections. The months of acute blood collection are presented for 97 samples submitted in 20132014 that were determined by active case detection or retrospectively to contain B. miyamotoi DNA. To provide a crude estimate of the incidence of BMD, we compared the frequency of PCR-confirmed findings of B. burgdorferi, B. miyamotoi, B. microti, and A. phagocytophilum in the patient samples submitted during the peak transmission months (May through October) in 2013 and 2014. Of 11515 unique samples that were submitted and tested for all 4 agents, 3.1% (exact binomial 95% CI, 2.7% to 3.4%) contained B. microti DNA, 1.4% (CI, 1.2% to 1.6%) contained A. phagocytophilum DNA, and 2.5% (CI, 2.2% to 2.8%) contained Borrelia species DNA. Borrelia miyamotoi DNA was detected in 0.8% (CI, 0.6% to 1.0%) of all samples; hence, 1.7% (CI, 1.5% to 1.9%) of all samples contained B. burgdorferi or cognate sequences that were not assigned to species. Clinical Spectrum of BMD Basic clinical data were available for 51 case patients with definitive BMD selected out of 97 total. The clinical spectrum of disease was variable, but presenting symptoms were often suggestive of an undifferentiated flu-like illness. Patients presented with acute headache, fever, and chills and were often found to have leukopenia, thrombocytopenia, and elevated aminotransferase levels, mimicking human anaplasmosis infection (Tables 1 and 2). Patients were commonly described as appearing toxic; more than 50% were suspected of having sepsis, and 24% required hospitalization. The headaches were most commonly described as severe, resulting in head computed tomography scans and spinal taps in 5 patients. Two patients presented with recurrent fever, and 1 of them, who was not treated initially, yielded blood samples drawn a month apart that were positive on PCR. Table 1. Clinical Features of the 51 Case Patients With BMD Table 2. Laboratory Findings for the 51 Case Patients With BMD Treatment regimens were typical for suspected tickborne disease in our referral area practices. Of the 51 case patients, 40 received a 2- to 4-week course of oral doxycycline. Seven of 51 received oral amoxicillin, and 3 of these had received 1 or 2 doses of ceftriaxone beforehand. One of 51 received levofloxacin for 10 days. One case patient first received atovaquone and azithromycin for a laboratory-confirmed diagnosis of babesiosis and later received ceftriaxone after the results of laboratory assays were reported. Of the 42 patients for whom information on outcome was available, 40 had prompt resolution of

Nonrandom Distribution of Vector Ticks (Dermacentor variabilis) Infected by Francisella tularensis

The island of Marthas Vineyard, Massachusetts, is the site of a sustained outbreak of tularemia due to Francisella tularensis tularensis. Dog ticks, Dermacentor variabilis, appear to be critical in the perpetuation of the agent there. Tularemia has long been characterized as an agent of natural focality, stably persisting in characteristic sites of transmission, but this suggestion has never been rigorously tested. Accordingly, we sought to identify a natural focus of transmission of the agent of tularemia by mapping the distribution of PCR-positive ticks. From 2004 to 2007, questing D. variabilis were collected from 85 individual waypoints along a 1.5 km transect in a field site on Marthas Vineyard. The positions of PCR-positive ticks were then mapped using ArcGIS. Cluster analysis identified an area approximately 290 meters in diameter, 9 waypoints, that was significantly more likely to yield PCR-positive ticks (relative risk 3.3, P = 0.001) than the rest of the field site. Genotyping of F. tularensis using variable number tandem repeat (VNTR) analysis on PCR-positive ticks yielded 13 different haplotypes, the vast majority of which was one dominant haplotype. Positive ticks collected in the cluster were 3.4 times (relative risk = 3.4, P<0.0001) more likely to have an uncommon haplotype than those collected elsewhere from the transect. We conclude that we have identified a microfocus where the agent of tularemia stably perpetuates and that this area is where genetic diversity is generated.

Genotypic diversity of Francisella tularensis infecting Dermacentor variabilis ticks on Martha's Vineyard, Massachusetts

ABSTRACT Marthas Vineyard, Mass., has been the site of two outbreaks of tularemia (1978 and 2000). Although most patients from both outbreaks presented with pneumonic disease and although aerosol transmission has been suggested, the bite of a dog tick and exposure to rabbits remain the only proven modes of transmission. The factors that precipitated the tularemia outbreaks or the proximal determinants of human risk remain undescribed. We sought to test the hypothesis that the ongoing outbreak is due to a recent introduction event as opposed to amplification of a cryptic enzootic cycle. From 2001-2003, we collected 4,246 dog ticks and tested them in pools for evidence of tularemia by PCR. We then measured the genetic diversity of Francisella tularensis by using multiple-locus variable-number tandem repeat analysis. The prevalence of F. tularensis in dog ticks averaged 0.7%. From 29 positive pools, we identified 10 unique genotypes, which was an unexpectedly large degree of diversity (Simpsons index = 0.86). This degree of genetic diversity is inconsistent with a recent introduction event. We conclude that there has been long-standing enzootic transmission of tularemia on the island.

Enzootic Transmission of Anaplasma bovis in Nantucket Cottontail Rabbits

ABSTRACT Serological studies of cottontail rabbits sampled from Nantucket Island, Mass., have suggested exposure to at least two ehrlichiae. The agent of human granulocytic ehrlichiosis (Anaplasma phagocytophilum) is intensely enzootic in rabbits there, but the identity of the other ehrlichial infection remains undescribed. We sampled rabbits over five transmission seasons and tested their blood and tissues for evidence of infection using PCR targeting an Ehrlichia genus-wide 16S rDNA target. Sequence analysis of positive amplicons revealed the presence of Anaplasma bovis, an agent not known to be present in North America. The average annual prevalence of A. bovis within rabbits, as determined by PCR of blood samples, was 18%. Haemaphysalis leporispalustris appears to serve as vector. The public health (human or veterinary) significance of this finding remains speculative.

Molecular Detection of Bartonella schoenbuchensis from Ectoparasites of Deer in Massachusetts

Deer keds (Lipoptena cervi) are thought to have been introduced into New England from Europe during the 1800 s. We sought to determine whether L. cervi from Massachusetts deer contained evidence of infection by Bartonella schoenbuchensis, which appears to be maintained by L. cervi in Europe. Five of 6 keds were found to contain B. schoenbuchensis DNA, and 2 deer ticks cofeeding on deer with such keds did as well. The detection of Bartonella DNA in deer ticks probably represents contamination by infected deer blood.

Rickettsia rickettsii (Rickettsiales: Rickettsiaceae) in Amblyomma americanum (Acari: Ixodidae) From Kansas

ABSTRACT The role of lone star ticks as vectors for Rocky Mountain spotted fever (RMSF) remains poorly described. We compared the entomological inoculation rates (EIRs) for Rickettsia spp. for representative sites in Missouri and Kansas, states that frequently report RMSF each year. Hostseeking ticks were collected during 2006 and pooled tick homogenates analyzed by polymerase chain reaction to detect probable R. rickettsii, with confirmation for multiple gene targets performed on individual ticks from pools that screened positive. Of 870 adult and nymphal lone star ticks, Amblyomma americanum (L.), 0.46% contained DNA of Rickettsia rickettsii. Interestingly, two of these positive ticks were concurrently infected by R. amblyommii. More than 90% of lone star tick pools contained R. amblyommii DNA. Of 169 dog ticks that were analyzed, none were infected by R. rickettsii. The entomological inoculation rate for spotted fever group (SFG) rickettsiae within lone star ticks was an order of magnitude greater than that for dog ticks. We conclude that lone star ticks may be epidemiologically significant vectors of Rocky Mountain spotted fever and of spotted fever group rickettsiae.

A new Francisella (Beggiatiales: Francisellaceae) inquiline within Dermacentor variabilis say (Acari: Ixodidae).

Abstract While estimating the prevalence of the Dermacentor variabilis (Say) symbiont (DVS) in dog ticks on Martha‘s Vineyard, MA, we identified DNA that may represent a heretofore unrecognized Francisella sp. Polymerase chain reaction targeting a portion of the 16S rDNA specific for DVS yielded an amplicon that was only 96.6% similar to that of DVS accessioned in GenBank. Phylogenetic analysis of the 16S and 23S rDNA genes suggests the presence of a distinct bacterium closely related to the other endosymbionts of Dermacentor spp. Fifty-five percent of dog ticks tested from three sites in Massachusetts showed evidence of infection with this new agent, called Dermacentor variabilis francisella (DVF), whereas 100% tested positive for DVS. All larval progeny of dog ticks known to contain DVF also showed evidence of colonization, demonstrating that this agent may be maintained by transovarial transmission. Coinfection of ticks with both Francisella species did not seem to interfere with transmission.

Prevalence of Ehrlichia muris in Wisconsin Deer Ticks Collected During the Mid 1990s.

Human ehrlichiosis is due to infection by tick transmitted bacteria of the genus Ehrlichia. Based on a hypothesis for the biogeography of deer tick transmitted infections, we undertook a focused search for the Eurasian E. muris in North American deer ticks. The search was stimulated by anecdotal reports of E. muris-like infection in human ehrlichiosis patients from Wisconsin. We analyzed archived adult deer ticks collected in northern Wisconsin during the 1990s by specific polymerase chain reaction for evidence of infection, and sequenced amplification products to identify E. muris. About 1% of 760 adult deer ticks collected from Spooner, Wisconsin in the 1990s contained E. muris DNA. We conclude that E. muris was present in North American deer ticks a decade ago and is likely to infect this human biting vector elsewhere in the U.S. Biogeographic theory and molecular phylogenetic methods can facilitate a targeted search for potential zoonoses.